屋尘螨第13组变应原克隆表达、纯化及免疫原性鉴定  

Cloning,expression,purification and immunogenicity identification of the recombinant Allergen Der p13 from Dermatophagoides pteronyssinus

作　　者：官秀丽 袁如意 黄妙琴 杨礼腾 刘志刚[1] 刘晓宇[1] GUAN Xiu-li;YUAN Ru-yi;HUANG Miao-qin;YANG Li-teng;LIU Zhi-gang;LIU Xiao-yu(Institute of Allergy and Immunology,Shenzhen University,Shenzhen,Guangdong 518060,China;Department of Allergy,The Third Affiliated Hospital of Shenzhen University,Shenzhen,Guangdong 518060,China)

机构地区：[1]深圳大学过敏反应与免疫学研究所,广东深圳518060 [2]深圳大学第三附属医院变态反应科,广东深圳518060

出　　处：《中国热带医学》2021年第4期303-307,共5页China Tropical Medicine

基　　金：国家自然科学基金(No.31729002,No.U1801286,No.81971514);广州市科学研究计划重点项目(No.201804020043);深圳市孔雀计划团队项目(No.KQTD20170331145453160);深圳市科技计划国际科技合作项目(No.GJHZ2018041819053);南山区“领航团队”支持计划项目(No.LHTD20180007);呼吸疾病国家重点实验室开放课题(No.SKLRD-OP-201909)。

摘　　要：目的克隆表达及纯化屋尘螨(Dermatophagoides pteronyssinus,Der p)第13组变应原(Der p13)蛋白,并鉴定其免疫原性。方法提取屋尘螨总RNA,通过逆转录聚合酶链式反应(RT-PCR)技术获得Der p13的cDNA片段。正确设计引物,利用已获得的cDNA片段进行PCR扩增,获得Der p13基因片段。将扩增产物连接到载体上并转入克隆菌Top10中,挑选阳性菌落,保菌取样送到上海生工生物有限公司进行测序。将测序正确的基因转入表达载体PET-24a中,经Bam H I、Xho I双酶切后用异丙基硫代半乳糖苷(IPTG)诱导表达和镍柱亲和层析法纯化重组蛋白Der p13。使用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)以及免疫印迹(Western Blot)鉴定纯化产物。结果成功构建表达了质粒PET-24a-Der p13,SDS-PAGE电泳结果显示上清可溶性蛋白表达量较多,分子质量约为13000。免疫印迹结果表明该蛋白可与尘螨过敏患者血清结合,具有一定免疫原性,重组蛋白Der p13三级结构预测表明其主要是β折叠,根据DNAStar抗原指数、亲水性、表面可及性、柔韧性等数据可以推导出蛋白Der p13抗原区,重组蛋白Der p13进化树分析结果显示屋尘螨与粉尘螨、疥、害嗜鳞螨及储存螨同源性较高。结论成功克隆表达、纯化出Der p13重组蛋白,该蛋白具有一定的免疫原性,为临床实验研究过敏性疾病的诊断与治疗奠定基础。Objective To clone,express and purify the 13 th group of allergen(Der p13)protein from Dermatophagoides pteronyssinus,and to identify its immunogenicity.Methods The total RNA of Dermatophagoides pteronyssinus was extractedand cDNA fragment of Der p13 was obtained by RT-PCR.The primers were designed correctly,and Der p13 gene was obtainedby PCR amplification using the obtained cDNA fragment.The amplification products were linked to the vector and transferredinto E.coli TOP10.The positive colonies were picked out and retained.The simples of the positive colonies were sent forsequencing.The recombinant protein Der p13 was purified by Ni column affinity chromatography and induced by IPTG.Thepurified products were identified by 12 alkyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting.Results pET-24 a-Der p13 was successfully constructed.SDS-PAGE showed that the expression of soluble protein in thesupernatant was high and the molecular weight was about 13000.The results of Western blotting showed that the protein couldbind to the sera of patients with dust mite allergy and had certain immunogenicity.According to the data about antigenic index,hydrophilicity plot,surface probability and flexible regions of DNA Star,the antigenic of Der p13 could be deduced.Thephylogenetic tree analysis of Der p13 showed that Dermatophagoides pteronyssinus had high homology with Dermatophagoides farina,Sarcoptes scabiei,Lepidoglyphus destructor and Acarus siro.Conclusion The recombinant protein of Der p13 wassuccessfully cloned,expressed and purified.The protein has some immunogenicity,which lays a foundation for clinicalexperimental study of the diagnosis and treatment of allergic diseases.

关 键 词：屋尘螨 第13组变应原 表达纯化 免疫原性 三级结构 

分 类 号：R384.4[医药卫生—医学寄生虫学]